MOLECULAR ANALYSIS OF MAMMALIAN KINETOCHORE STRUCTURE

复制的基因组准确分离成子代 处于有丝分裂和减数分裂的细胞是 多细胞生物的正常生长和分化。 不适当的染色体分离直接与人类 发育障碍，特别是唐氏综合症。这项工作 在这里提出的是为了详细阐明分子 人类动粒的组成，即染色体结构 直接负责将染色体与有丝分裂相联系 隔离装置。 CREST患者的自身免疫性疾病血清 硬皮病已经被证明识别三种特定的 动粒结合蛋白存在于人类染色体上， 称为CENP-A、CENP-C。这项工作的一个主要目标已经 部分实现了，是使用CREST自身抗体来分离 利用生物信息学技术克隆CENPs编码基因 表达载体克隆。这些的主要结构 蛋白质将通过DNA测序来确定，有可能 提供对动粒结构和功能的洞察。CENP 蛋白质表达技术将被开发用于直接 CENP的分析：DNA相互作用和产生定义的 CENP功能精细分析的免疫学试剂。 第二个主要目标是使用抗CENP抗体直接 分离动粒颗粒。从纯化的DNA中提取DNA 动点(KDNA)将被用来建立重组DNA 用于克隆和分析这些重要基因的文库 序列。将开发分析CENP：DNA的分析方法 体外结合作用及kDNA：CENP-的结构 含有染色质的物质将在体外和体内进行研究 使用非洲爪哇卵母细胞分析系统。动粒结构和 在哺乳动物细胞中的功能将通过重新引入 CENP编码序列显微注射干扰转染 或者取消正常的CENP表达，分析改变的有丝分裂 细胞表型。KDNA序列在染色体中的作用 将通过开发以下系统来探索隔离 染色体外染色体动粒功能的重建 通过潜在的颠覆性的kDNA扩增。 这些实验将建立详细的工具和方法 人类染色体机制的分子分析 种族隔离。

The accurate segregation of the duplicated genome into daughter cells at mitosis and meiosis is a critically important component of the normal growth and differentiation of multicellular organisms. Improper chromosome segregation is directly linked to human developmental disorders, notably Down's syndrome. The work proposed here is designed to elucidate in detail the molecular components of the human kinetochore, the chromosomal structure that is directly responsible for linking chromosomes to the mitotic segregation apparatus. Autoimmune disease sera from patients suffering CREST scleroderma have been shown to recognize three specific kinetochore binding proteins present on human chromosomes, termed CENP-A, CENP-C. One major goal of this work, already partially realized, is to use CREST autoantibodies to isolated cDNA clones encoding the CENPs using the techniques of expression vector cloning. The primary structures of these proteins will be determined by DNA sequencing, potentially providing insight into kinetochore structure and function. CENP protein expression techniques will be developed for use in direct analysis of CENP:DNA interactions and to generate defined immmunological reagents for refined analysis of CENP function. A second major goal is to use anti-CENP antibodies to directly isolate kinetochore particles. DNA isolated from purified kinetochores (kDNA) will be used to establish recombinant DNA libraries for the cloning and analysis of these important sequences. Assays will be developed to analyze CENP:DNA binding interactions in vitro and the structure of kDNA:CENP- containing chromatin will be investigated in vitro and in vivo using Xenopus oocyte assay systems. Kinetochore structure and function will be probed in mammalian cells by re-introduction of CENP coding sequences microinjection of transfection to disrupt or abolish normal CENP expression, analyzing the altered mitotic phenotypes of cells. The role of kDNA sequences in chromosome segregation will be explored by developing systems for reconstitution of kinetochore function on extrachromosomal plasmids and by potentially disruptive amplification of kDNA. These experiments will establish tools and methods for detailed molecular analysis of the mechanisms of human chromosome segregation.